Ion transporting ATPases create ion gradients across the membranes of most eukaryotic and of some prokaryotic cells using the energy from ATP hydrolysis. E1-E2 ATPases are essential for maintaining the proper ionic composition of the eukaryotic cells. The E1-E2 designation refers to the fact that the enzyme can exist in two different states (E1 and E2) as it undergoes cycles of phosphorylation and dephosphorylation. Dysfunction of the regulatory processes involved in E1-E2 ATPase ion transport occur in many disease states arising infection, stress, and genetic factors. Furthermore, mammalian Na+, K+- ATPases are the primary site of action for the cardiotonic glycosides (digitalis glycosides) used in the treatment of heart failure. Unfortunately, many mammalian E1-E2 ATPases do not lend themselves easily to a biophysical analysis of their structure and function due to the heterogeneity and polydispersity of the protein preparations and their limited stability in detergent solutions. The K+-transporting Kdp ATPase from E. coli can now be obtained pure and in the amounts necessary for crystallization experiments. This large complex ( 285 KDa) contains three independent polypeptides, one of which (KdpB) has striking sequence similarities to the mammalian cell membrane Na+, K+- ATPase and Ca+-ATPase from the sarcoplasmic reticulum. The similarities in mechanism as well as this sequence homology with other E1-E2 ATPase suggest that this bacterial E1-E2 ATPase is a suitable model for the mammalian systems. Experiments will be undertaken to crystallize the Kdp-ATPase from detergent solutions. Methods developed by the principal investigator have successfully yielded crystals of other membrane proteins. The long term goal is to obtain large crystals of Kdp-ATPase suitable for a high resolution X-ray diffraction analysis.